Copper is an essential but toxic trace metal that has been implicated in oxidative damage to DNA, proteins, and membranes. The long-term goal of the proposed research is to better understand the mechanisms of copper-induced oxidative stress, and the molecular controls that organisms have developed to defend against oxidative damage. The blue crab, Callinectes sapidus, with its highly-active copper metabolism, is proposed as a nonmammalian model. The immediate goal is to establish the usefulness of the model by measureing baseline data on oxidative stress parameters in the blue crab. Recent work has shown that the intracellular antioxidant glutathione (GSH) in the hepatopancreas of marine crustaceans is involved in the metabolism of copper. GSH reduces Cu(II) to Cu(I), which results in the oxidation of GSH. Cu(I) forms a complex with reduced GSH, which may serve to deliver copper for the biosynthesis of the copper-dependent protein hemocyanin.The presence of three copper-inducible metallothionein genes in the hepatopancreas have also been demonstrated. Through its oxidation of GSH and its catalysis of superoxide and hydroxyl radical formation, copper may play a crucial role in the generation of oxidative stress. On the other hand, copper plays an important role in antioxidant defense by virtue of its catalytic role in the dismutation of superoxide by the enzyme Cu,Zn superoxide dismutase. Furthermore, metallothioneins have been implicated in protection against oxidative stress. These data suggest intricate, but as yet unresolved, relations between copper, GSH, metallothionein, and antioxidant enzymes. The objective of this proposal is to further explore and clarify these relationships. The proposed studies will include exposure of blue crabs and their hepatopancreas tissues to copper and the GSH-depleting agent, buthionine sulfoximine, and measuring the effect of these treatments on: (1) intracellular distribution of copper; (2) amounts of intracellular reduced and oxidized GSH; (3) activities of superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase, and (4) oxidative tissue damage as measured by the degree of lipid peroxidation. These pilot studies are designed to demonstrate the intricate linkages between copper, GSH, metallothionein, antioxidant enzymes and oxidative damage. The data will provide the basis for long-term studies that will investigate the molecular mechanisms that underlie these linkages, and will help to develop a nonmammalian model for the human disease that are caused or exacerbated by copper-mediated oxidative damage.